Gene regulation by ToxR, TcpP and ToxT in V. cholerae The major virulence factors of Vibrio cholerae, cholera toxin and toxin-coregulated pilus (CT and TCP, respectively) are controlled by a cascade of regulators that ultimately leads to activation of ToxT by two membrane-localized activators, ToxR and TcpP. ToxT is the direct activator of cholera toxin and toxin-coregulated pilus gene expression, as well as expression of other genes whose roles in the biology and pathogenicity of V. cholerae are less well defined. The membrane activators, ToxR and TcpP, each depend for their activity on a cognate effector protein, ToxS and TcpH, although the mechanism of this dependence may be different for each pair of proteins. A major discovery of the prior funding period is that TcpP is subjected to regulate intramembrane proteolysis that is antagonized by TcpH. This process requires the YaeL protease as the second of two proteases that eliminate TcpP from the cell. TcpP is necessary to activate the toxT gene, and another major discovery from the most recent period of NIH support for this work was identification of a small RNA molecule (tarA) under direct transcription control of ToxT. Based on significant preliminary data and published manuscripts, the current proposal has three aims: 1. Comprehensive analysis of TcpP and TcpH interaction and the regulated intramembrane proteolysis pathway that governs TcpP levels in the cell, including the identification of the still unknown site I protease; 2. Screens for small molecule inhibitors of the ToxR/TcpP arm of the regulatory pathway and characterization of the targets of those inhibitors as well as the mechanism of inhibitor; 3, the tarA mechanism of action. PUBLIC HEALTH RELEVANCE: Vibrio cholerae is a bacterium that causes the human diarrheal disease cholera. The proposed studies are designed to uncover new knowledge mechanisms used by V. cholerae to regulate its virulence traits. One of the aims is intended to identify small molecules that interfere with key steps in the regulation pathway leading to disease. Such molecules might represent new classes of drugs that target virulence.